The Study On The Regulation Of MiR-218 On Prostate Cancer Stem Cells And Its Molecular Mechanism

Prostate cancer(Pca)is one of the common malignant afflicting men.The current treatment of prostate cancer failed to achieve the desired effect.Castration resistant prostate cancer and tumor metastasis is a thorny problem.To explore the biological characteristics of prostate cancer and find potential therapeutic targets may be developed for clinical treatment.In recent years,studies have shown that cancer stem cells(CSCs)play an important role in local infiltration and metastasis of malignant tumors.CSCs are tumor cell progenitor cells,which account for very few parts of the tumor,but with similar to the adult stem cell self-renewal ability and differentiation potential,is the tumor occurrence,proliferation,recurrence and other processes of the starting cell.To maintain the tumor within the steady state and the invasion of the tumor increased and then play a decisive role in the transfer.MiRNAs(microRN As)are a class of endogenous small non-coding RNAs,called micro RNAs,consisting of about 22 nucleotide molecules that block the translation of target gene mRNA by binding to the 3 'UTR of the target gene mRN A,Expression of negative regulatory genes.It has been studied that they are in the development and progression of human cancer play an important role in the regulation of molecules.The latest evidence suggests that abnormal expression of miRNAs is associated with a large number of cancer stem cell disorders in cancerous diseases.The latest evidence that the aberrant expression of miRNAs and cancer stem cell disorder in a large number of cancer diseases.MiR-218 research in recent years has become a hot,and lung cancer,nasopharyngeal cancer,bladder cancer,breast cancer and other malignancies,in different tumor regulation of target genes and there are different kinds of change.RELN(reelin)is an extracellular glycoprotein,is composed of 3,461 amino acid composition of extracellular matrix proteins,and neuronal cells and several types of non-neuronal cell positioning and migration-related.New research shows that RELN expression in the cells of multiple myeloma,and RELN expression associated with poor prognosis myeloma cells.Reelin in cell migration and role in cancer metastasis,to a large extent is not clear,particularly in cancer stem cells(CSCs)role is not clear.Previous studies have shown that miR-218 is abnormally expressed in prostate cancer tissues.However,the exact mechanism of action of miR-218 is not clear whether the regulation of CSCs and the biological behavior of prostate cancer and how to regulate CSCs are not elucidated.Therefore,in-depth study of miR-218 abnormal expression in prostate cancer stem cells will help us to understand the invasion and metastasis of prostate cancer biological behavior and its potential mechanism.It'll provide new indicators for the early diagnosis and prognosis of prostate cancer.In this thesis,we study that miR-218 influence prostate cance r stem cells through the downstream target gene RELN.Objective:(1)The role of miR-218 in prostate cancer and prostate cancer stem cells was explored to lay the foundation for further study of its function in prostate cancer stem cells.(2)To investigate the role of miR-218 in CD44 + / CD133 + PC-3CSCs.(3)To investigate the effect of miR-218 and RELN on the proliferation and invasion of prostate cancer cells,and to clarify the function of target gene RELN.Methods:(1)The expression of miR-218 in 40 cases of human prostate cancer tissues and their adjacent tissues was detected by quantitative real-time PCR(qRT-PCR).(2)CD44 + / CD133 + prostate cancer stem cells and non-stem cell populations were isolated from prostate cancer cells by immunomagnetic beads.The CSCs populations was detected by flow cytometry.The expression of miR-218 in the two cell populations were detected by q RT-PCR.(3)MiR-218 mimics was transfected into prostate cancer stem cell PC-3 CSCs to construct miR-218 overexpressing cell line.QRT-PCR was used to verify the expression of miR-218.(4)The effect of miR-218 on the proliferation of PC-3 CSCs was detected by MTT assay.The effect of miR-218 on the clone formation ability of PC-3 CSCs was detected by plate clone formation assay.The tumor cell formation test was used to test the self-renewal ability of PC-3 CSCs.The effect of miR-218 on the invasion and metastasis of PC-3 CSCs was detected by Transwell invasion assay.The effect of miR-218 on the tumorigenic ability of PC-3 CSCs was detected by nude mice tumorigenesis assay.(5)The target gene of tumor metastasis was screened by TargetScan,picTar and miRnada target gene prediction software.We conducted a luciferase reporter assay to confirm whether RELN was a direct target of miR-218.The expression of RELN protein was detected by Western blot in miR-218 mimics,inhibitor and NC.(6)After transfection of RELN overexpressing vector into miR-218 CSCs cells,the effect of RELN on the proliferation of miR-218 CSCs was detected by MTT assay.The effect of RELN on the cloning ability of miR-218 CSCs was detected by plate cloning assay.The effect of RELN on the invasion and metastasis of miR-218 CSCs was detected by Transwell invasion assay.The effect of RELN on the tumorigenic ability of miR-218 CSCs was detected by nude mice tumorigenesis assay.Results:(1)The expression of miR-218 in prostate cancer tissues was significantly lower than that in adjacent tissues(P <0.01).(2)The results of flow cytometry showed that CD44 + / CD133 + cells were 98.2% in the prostate cancer stem cell group selected by immunomagnetic sorting method.The results of q RT-PCR showed that the expression of miR-218 in prostate cancer stem cells was significantly lower than that in non-prostate cancer stem cells(P <0.01).(3)The results of q RT-PCR showed that miR-218 expression in miR-218 mimics cells was nearly 20-fold higher than that in control miR-NC group(p <0.01).(4)MTT assay showed that the cell proliferation ability of miR-218 mimics group was significantly lower than that of miR-218 group(p <0.01).The results of plate clone formation showed that the miR-218 mimics group was cloned(P <0.01).Compared with the miR-NC group,the miN-218 mimics group had a significant reduction in the ball-ball ability(p <0.01).The results of the Transwell invasion assay showed that the miR-(P <0.01).The results of nude mice tumorigenesis assay showed that the tumorigenic ability of miR-218 mimics group was significantly lower than that of miR-218 mimics group(P <0.01).(5)The results of luciferase reporter assay showed that the fluorescence intensity of wild type group was significantly decreased(p<0.01),but there was no significant change in mutant group.The results of Western blot showed that,compared with group miR-NC,the RELN protein was decreased in miR-218 mimics group,but increased obviously in miR-218 inhibitor group.(6)MTT assay showed that the cell proliferation ability of miR-218 + RELN group was significantly higher than that of miR-218 group(p <0.01).The results of plate clone formation showed that the clone formation ability of miR-218 + RELN group was significantly higher than that of miR-218 group(p <0.01).Transwell invasion assay showed that the migration ability of miR-218 + RELN group was significantly higher than that of miR-218 group(p <0.01).The results of nude mice tumorigenesis assay showed that the tumorigenic ability of miR-218 + RELN cells was significantly higher than that of miR-218 group(p <0.01).Conclusion:(1)Immune magnetic bead sorting method can successfully sort out prostate cancer stem cells.(2)MiR-218 is low in prostate cancer and prostate cancer stem cells.(3)MiR-218 can inhibit proliferation,clonal formation,ball ability,invasive ability and in vivo tumorigenic ability of PC-3 CSCs.MiR-218 inhibits stemness of PC-3CSCs.(4)MiR-218 can inhibit the expression of RELN protein by binding RELN 3'UTR.MiR-218 can regulate the stemness of PC-3CSCs by inhibiting the expression of RELN.